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ORIGINAL ARTICLE
Year : 2014  |  Volume : 3  |  Issue : 3  |  Page : 141-146

Validated HPTLC technique for simultaneous estimation of candesertan celexitil and hydrochlorothiazide in pharmaceutical dosage form


1 Department of Pharmaceutical Analysis, Jam'iyyat Da'wa Tablighul Islam College of Pharmacy, Calicut, Kerala, India
2 Department of Pharmaceutical Chemistry, Nitte Gulabi Shetty Memorial Institute of Pharmaceutical Sciences, Nitte University, Deralakatte, Mangalore, Karnataka, India

Date of Web Publication7-Oct-2014

Correspondence Address:
Sarif Niroushkonari
Department of Pharmaceutical Analysis, Jam'iyyat Da'wa Tablighul Islam College of Pharmacy, Calicut - 673 012, Kerala
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/2278-0521.142320

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  Abstract 

Aim: Analytical HPTLC technique was developed for the simultaneous estimation of candesertan celexitil (CAN) and hydrochlorothiazide (HYD) in 2 totally different strengths of pharmaceutical dosage form. Materials and Methods: Chromatography was performed on 60F 254 TLC pre-coated aluminum plates with mobile phase of toluene: ethyl acetate: formic acid (85%) in the ratio of 6:4:1 v/v. The mean Rf values with standard deviation were found to be 0.39 ± 0.01 and 0.73 ± 0.01 for CAN and HYD, respectively. Results: The linear regression data has shown a good linear relationship over a concentration range of 200-1200 ng/spot for each drug with a correlation coefficient of 0.9999. Limit of quantification and the limit of detection establish the sensitivity of the developed technique. The stability study indicates that drugs were stable up to 72 hours. The technique was precise and relative standard deviations were found to be less than 0.05%. System suitability tests are used to verify that the resolution and repeatability of the system is adequate for the analysis. The technique was specific and selective since there is no additional peak beside with main peak. The Robustness study, recovery study and percentage of assay of the formulation were within the limit as per ICH guidelines. Conclusion: A simple, rapid, cost-effective, eco-friendly, specific, precise, sensitive and efficient analytical HPTLC technique was developed for the simultaneous estimation of candesertan celexitil (CAN) and hydrochlorothiazide (HYD) completely in 2 totally different strength of pharmaceutical marketed formulations.

Keywords: Candesertan celexitil, hydrochlorothiazide, high performance thin layer liquid chromatography, ICH-guidelines, simultaneous method


How to cite this article:
Niroushkonari S, Jacob JT. Validated HPTLC technique for simultaneous estimation of candesertan celexitil and hydrochlorothiazide in pharmaceutical dosage form. Saudi J Health Sci 2014;3:141-6

How to cite this URL:
Niroushkonari S, Jacob JT. Validated HPTLC technique for simultaneous estimation of candesertan celexitil and hydrochlorothiazide in pharmaceutical dosage form. Saudi J Health Sci [serial online] 2014 [cited 2021 May 15];3:141-6. Available from: https://www.saudijhealthsci.org/text.asp?2014/3/3/141/142320


  Introduction Top


Candesertan celexitil (CAN) chemically [1] is1-{[(Cyclohexyloxy) carbonyl] oxy} ethyl 2-ethoxy-1-{[2'-(1H-tetrazol-5-yl)-4-biphenylyl] methyl}-1H-benzimidazole-7-carboxylate. It is an angiotensin II type-1 receptor antagonist used as an antihypertensive. Hydrochlorothiazide (HYD) chemically [2] is s6-chloro-1, 1-dioxo-3, 4-dihydro-2H-1, 2, 4-benzothiadiazine-7 sulfonamide. It is a thiazide diuretic used for treatment of hypertension, heart failure, andrenal and hepatic disorders. When monotherapy encountered bottlenecks in treating cardiovascular disease, fixed-dose combos were suggested as a good and safe program for initiating medical care particularly for the patients with complications. Each drug in perfect combination will exert its best effectiveness with fewer side effects. Additionally, fixed-dose combinations also brought economic benefits for patients with fewer medications compare with several drugs administered separately. Based on the clinical practice and market research, fixed-dose formulations are becoming a promising choice for hypertensive patients gradually. [3]

HPTLC has become a routine analytical technique due to its benefits of reliability in quantitation of analytes at micro and even in nanogram levels and cost-effectiveness. The key advantage of HPTLC is that many samples will be analyzed at the same time, employing a little amount of mobile phase unlike HPLC. This can effectively cut back the time and cost of drug analysis. Extensive literature reviews revealed single simultaneous estimation [4] and another lone analysis [5] of both CAN and HYD using HPTLC. HPTLC method of CAN with another drug combination was also reported. [6] Literature review also reported few spectrophotometric and RP-HPLC methods for the analysis of CAN and HYD. [7],[8],[9],[10],[11],[12] Literature review also pointed out that all existing methods are sensitive only up to the microgram level or require large amounts of organic solvents, which may be expensive in the determination of both the drugs in routine analysis. The objective of the present study was to develop a cost-effective HTPLC technique for the simultaneous determination of CAN and HYD in pharmaceutical dosage forms.


  Materials and methods Top


Chemicals and reagents

The drug samples were obtained as a gift from Mylan laboratories limited, Hyderabad, India. All the solvents used in the method development were of analytical grades. Silica gel 60F 254 TLC pre-coated aluminum plates (10 × 10 cm, layer thickness 0.2 mm, E. Merck, Mumbai) were used as the stationary phase. HPLC-grade methanol, ethyl acetate, chloroform, toluene, hexane and formic acid (85%) were procured from Merck India. Formulation containing CAN and HYD (Candesar-H tablets; Ranbaxy India and Candelong-H; Micro Labs' Cardicare) were purchased from resident pharmaceutical shop.

Instrumentation and chromatographic conditions

Instruments used in the study were CAMAG HPTLC: A conventional CAMAG 20 × 10 cm twin-trough chamber and ultra sonicator were used for the development of chromatogram. Automatic TLC Sampler 4 was used as sample applicator with Hamilton syringe; CAMAG TLC Scanner 3 was used for scanning the chromatogram with Wincats Software. The experiment was performed on 60F 254 TLC pre-coated aluminum sheets (10× 10 cm, layer 0.2 mm thickness, E. Merck, Mumbai), which were used as stationary phase and it was previously prewashed using methanol. Samples were spotted at a speed of 10/mms to the plates as 7 mm bands, 0.5 mm apart, and 10.5 mm from the edges of the plate and 10 mm from the bottom of the plate. The plates were developed by the ascending technique, to a total distance of 90 mm, at 25 ± 5°C, relative humidity 50-60%, in a CAMAG twin-trough glass chamber with a stainless steel lid, using a mobile phase of toluene:ethyl acetate:formic acid (85%) in the ratio of 6:4:1 v/v and the chamber saturation time of 20 minutes. After development of plates, it was dried and then viewed in a CAMAG UV cabinet. After 10 minutes, it was subjected for scanning with a CAMAG TLC Scanner 3, using Wincats software in absorbance mode, with slit dimensions 6.00 × 0.2 mm (Micro). The detection of wavelengths was selected at 254 nm by overlain spectra of the drugs acquired in situ from the plate. The Mean Rf values with standard deviation were established at 0.39 ± 0.01 and 0.73 ± 0.01 for CAN and HYD, respectively.

Preparation of calibration curves

Standard stock solutions of CAN and HYD were prepared by dissolving 10 mg standard drugs in 10 ml of standard flask by using diluents to get a concentration of 1000 μg/ml of CAN and HYD separately (stock solution). From the above stock solutions of 0.2, 0.4, 0.6, 0.8, 1 and1.2 μL of CAN and HYD was spotted on the pre-coated silica gel 60F 254 TLC aluminum plates. The plate was dried, developed and analyzed densitometrically. The linear regression data showed a good linear relationship over a concentration range of 200-1200 ng/spot for both drugs.

Analysis of formulations

Twenty tablets containing CAN and HYD of different strengths were taken and its powder equivalent to 10 mg was weighed. Then, it was diluted in a 10 ml standard flask to get concentration of 1000 μg/ml by using diluents. The resulting solutions were sonicated for 20 minutes and filtered through 0.45 μ Whatman filter paper No. 41 (Sigma-Aldrich). From the above stock solutions, 0.4 μL was spotted for brand A and for brand B, it was 0.8 μL.

Validation of chromatographic technique

Accuracy or recovery

To confirm the accuracy of the projected method, recovery experiments were performed by standard addition technique by adding a known amount of standards to the sample at three different levels (80%, 100%, 120%). Each levels were repeated three times (n = 3). This solution was spotted on the plate followed by development and scanning three times.

Precision

Precision is the measure of how close the data values are to each other for a number of measurements under the same analytical conditions. It was performed by using a concentration of 200 ng/spot for HYD and 400 ng/spot for CAN and repeated three times (n = 3).

Calibration, linearity and range

Linearity curves were demonstrated by preparing and analyzing the standard preparation of six completely different concentrations of each drug, ranging from 200-1200 ng/spot. From this experiment, coefficient of correlations, intercept and slope was calculated.

Limits of detection and quantification

The sensitivity of measurement was estimated in terms of the limit of quantification (LOQ) and the limit of detection (LOD). LOQ and LOD were calculated by the use of equations LOD = 3 × (N/B) and LOQ = 10 × (N/B) where N is the standard deviation of the peak area of the drugs (n = 6), taken as a measure of noise and B is the slope of the corresponding calibration plot.

Stability studies

Stability studies were carried out on the chromatographic plate for different period. The analyte was tested against freshly prepared standard solutions and the developed plates were scanned at different intervals of time, such as 6, 12, 24 and 48 hours.

Specificity and selectivity

Rf values of standard and drug formulations demonstrate the specificity of method. LOQ and LOD parameters establish sensitivity in nanogram level of the developed technique.

System-suitability study

System-suitability tests are used to verify that the resolution and repeatability of the system is adequate for the analysis. System suitability was assessed by replicate (n = 6) analysis of a mixed standard solution of CAN and HYD under the conditions described above. Spots of 0.4 μL of system suitability solution in six replicates were noted.

Robustness and ruggedness

Robustness of the method was performed by spotting 0.4 μL and 0.8 μL of formulation from 1000 μg/mL formulation stock solution. It was conducted by making small deliberate changes in various optimized chromatographic conditions, and changes were made on composition of the mobile phase, duration of saturation time, development distance and time from development of plate to scanning. Ruggedness of the method or inter-day precision was performed on the drug on different days by maintaining the same experimental and environmental conditions.


  Result and discussion Top


Depending upon the nature of the two drug molecules, it absolutely was set to undertake numerous sorts of solvents such as hexane: acetone (6:4), toluene: ethyl acteate: methanol (6:4:0.2), toluene:chloroform:methanol (7:3:0.5). [12],[13],[14],[15],[16] The above trials failed to produce ideal results since the spots either moved along with the solvent front or gave split peaks for not at all moving from the applied site. Then, toluene: ethyl acetate: formic acid (85%) (6:4:0.5) was tried to give dense compact peak with less resolve. Finally, toluene:ethyl acetate: formic acid (85%) (6:4:1) gave dense and compact spots (with appropriate and significantly well-separated R f values of 0.39 ± 0.01 and 0.73 ± 0.01 for CAN and HYD, respectively).

The established technique, which is widely compatible for the estimation of different brands and strength of marketed formulation such as Candelong-H and Candesar-H, was simultaneously estimated to be in between 99% and 100%. Results are presented in [Figure 1] and [Figure 2] and [Table 1].
Figure 1: Chromatogram of formulation brand A

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Figure 2: Chromatogram of formulation brand B

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Table 1: Analysis of formulation

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Results and statistical parameters [17] of the recovery study established that it falls within ICH [18] limits. Mean recovery results of CAN and HYD were 100.05% and 100.22%, respectively [Table 2]. LOQ and the LOD parameters point out the sensitivity of developed technique because it was estimated in nanogram level. [4],[5],[18] LOD was found at 50.90 and 55.50 ng/spot for CAN and HYD, respectively. LOQ was found at 154.26, 185.006 ng/spot for CAN and HYD, respectively.
Table 2: Recovery study

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Precision of the analytical technique was established for interday (ruggedness), intraday and repeatability method/procedure. The percent RSD was calculated and is shown in [Table 3]. Calibration curve establishes outstanding linear relationship of six completely different concentrations of each drugs ranging from 200-1200 ng/spot. From this experiment, coefficient of correlation (0.999) with regression equation y = 12.319×-24.56 for CAN y = 10.844×-12.011 for HYD was calculated. [17] The results are shown in [Figure 3],[Figure 4],[Figure 5] and [Figure 6].
Figure 3: 3D chromatogram of standard solution of candesertan celexitil

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Figure 4: Calibration curve of candesertan celexitil

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Figure 5: 3D chromatogram of standard solution of hydrochlorothiazide

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Figure 6: Calibration curve of hydrochlorothiazide

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Table 3: Precision study

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During stability study of developed technique, there was no major deviation in the peak area (RSD <2.5%) of up to 72 hours. Drug decomposition was not observed throughout the chromatogram development. These interpretations suggest that the drug is stable under the typical processing and storage conditions of the analytical procedure. Specificity of the developed technique was confirmed by comparing Rf values of the spot with that of the standard, and there was no additional peaks observed for sample drug formulation. In system suitability study, RSD of peak areas of CAN and HYD was 0.05% to point out that the method was highly suitable for the routine analysis of both drugs. In addition to it, robustness studies were conducted for five different parameters indicating that method does not effect by making small deliberate changes in various optimized chromatographic conditions. Results are summarized in [Table 4]. [4],[5] In the developed technique, the dimension of the plates used was 10 × 10 cm, whereas in the reported method it was in the dimension of 20 × 10 cm. [4],[5] Furthermore, mobile used was cheap and easily available. Consumption of mobile phase to develop chromatogram is very less in contrast to RP-HPLC method, [7],[8],[9],[10] so the developed technique was cost-effective in terms of solvent consumption and plate dimension used in the analysis.
Table 4: Robustness study

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The validation of the developed method as per ICH guidelines [18] indicates that the technique was highly reproducible, extremely precise, rapid, simple, economical, sensitive, accurate, stable and specific for simultaneous estimation of CAN and HYD in pharmaceutical combined dosage form, so that it may be effectively applied for routine analysis in research institutions, quality control department in industries and for approved testing laboratories.


  Acknowledgments Top


The authors are grateful to Mylan laboratories limited, Hyderabad for providing the gift sample and to Nitte University Mangalore, Karnataka State, India for providing other necessary facilities to carry out this research work. The authors are also very thankful to department of drug standardisation laboratory, centre for medicinal plant research, Arya vaidya sala, kottakkal for providing necessary lab facilities and to Mr. Sreekanth Nadiq, manager- analytical R and D of Mylan laboratories limited, Hyderabad for their continuous encouragement to carry out this project.

 
  References Top

1.The Merck Index, 14 th ed. Whitehouse Station, NJ: Merck Research Laboratories Division of Merck and Co. Inc.; 2006. p. 1742.  Back to cited text no. 1
    
2.The Merck Index, 14 th ed. Whitehouse Station, NJ: Merck Research Laboratories Division of Merck and Co. Inc.; 2006. p. 4785.  Back to cited text no. 2
    
3.Xinhuan W, Panqin M, Xiangrong Z. A promising choice in hypertension treatment: Fixed-dose combinations. Asian J Pharm Sci 2014;9:1-7.  Back to cited text no. 3
    
4.Youssefa RM, Mahera HM, Hassana EM, El-Kimarya EI, Bararya MA. Development and validation of HPTLC and spectrophotometric methods for simultaneous determination of Candesartan Cilexetil and hydrochlorothiazide in pharmaceutical preparation. Int J Appl Chem 2010;6:233-46.  Back to cited text no. 4
    
5.Mehta BH, Morge SB. HPTLC-densitometric analysis of candesartan cilexetiland hydrochlorothiazide in tablets. J Planar Chromatogr 2008;21:173-6.  Back to cited text no. 5
    
6.Kondawar M, Gaikwad R, Apate V, Ravetkar A. High Performance Thin Layer Chromatographic determination of Enalapril maleate Hydrochlorothiazide in Pharmaceutical dosage form. Int J Pharm Tech Res 2011;3:1454-8.  Back to cited text no. 6
    
7.Qutab SS, Razzaq SN, Ashfaq M, Shuja ZA, Khan IU. Simple and sensitive LC-UV method for simultaneous analysis of hydrochlorothiazide and candesartan cilexetil in pharmaceutical formulations. Acta Chromatogr 2007;19:119-29.  Back to cited text no. 7
    
8.Balamuralikrishna K, Syamasundar B. Development and validation of high performance liquid chromatographic method for the Simultaneous Estimation of Candesartan cilexetil and Hydrochlorothiazide in combined tablet dosage form. Der Pharm Chem 2010;2:231-7.  Back to cited text no. 8
    
9.Narendra D, Satyanarayana T, Ganga Rao B. Simultaneous determination of Candesartan and Hydrochlorothiazide in combined Pharmaceutical Dosage form by New RP-HPLC Method. Res J Pharm Biol Chem Sci 2012;3:270-8.  Back to cited text no. 9
    
10.Annapurna M, Narendra K, Ravi Kumar. Liquid chromatographic method for the simultaneous quantitative determination of candesartan cilexetil and hydrochlorthiazide in pharmaceutical dosage forms. J Drug Deliv Ther 2012;2:48-54.  Back to cited text no. 10
    
11.Patel J, Dave JB, Patel CN, Patel D. Q-analysis Spectrophotometric methods for estimation of candesartan cilexetil and hydrochlorothiazide in tablet dosage form. J Chem Pharm Res 2010;2:10-4.  Back to cited text no. 11
    
12.Jacob JT, Aghera JP, Joshi CK. Analytical methods for the estimation of Candesartan in Pharmaceutical Formulations. J Pharm Res 2011;4:3930-2.  Back to cited text no. 12
    
13.Konari SN, Jacob JT. Stability-indicating LC-analytical method development and validation for the simultaneous estimation of flucloxacillin and amoxicillin in pharmaceutical dosage form. J Taibah Univ Sci 2014; http://dx.doi.org/10.1016/j.jtusci.2014.07.005.  Back to cited text no. 13
    
14.Niroushkonari S, Jacob JT. Development and validation of RP-HPLC method for the simultaneous estimation of prasugrel and aspirin in bulk and pharmaceutical dosage form. Inventi impact: Pharm Anal Qual Assur 2013;1:71-4.  Back to cited text no. 14
    
15.Niroushkonari S, Jacob JT. Stability indicating isocratic RP-HPLC DAD method for the simultaneous estimation of flucloxacillin and ampicillin in pharmaceutical dosage form: Application for routine drug analysis in quality control laboratories and clinical pharmacokinetic studies. Inventi impact: Pharm Anal Qual Assur 2014;3:158-64.  Back to cited text no. 15
    
16.Satinder A, Stephen S. Handbook of Modern Pharmaceutical Analysis, Separation Science and Technology Series. New York: Academic Press; 2001. p. 346-50.  Back to cited text no. 16
    
17.Charles HB, Corrinne P. Understandable statistics. Lexington, Toronto, Canada: D.C. Heath and Company; 1987. p. 390-5.  Back to cited text no. 17
    
18.ICH Guidelines Q2(R1) (2005) Validation of analytical procedures: Text and methodology. Available from: http://www.ich.org/fileadmin/Public_Web_Site/ICH_Products/Guidelines/Quality/Q2_R1/Step4/Q2_R1__Guideline.pdf. [Last accessed on 2011 Mar 10].  Back to cited text no. 18
    


    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6]
 
 
    Tables

  [Table 1], [Table 2], [Table 3], [Table 4]


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